本文選題：蛇毒 + AHV-PI��； 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:觀察(AHV-PI)對小鼠的急性毒性作用,探討該組分相關(guān)的理化性質(zhì)。方法:1.采用陰陽離子交換法和分子篩蛇毒蛋白純化柱純化皖南蝮蛇粗毒,純化后通過抑制血小板聚集來篩選目的峰即為AHV-PI,將目的峰凍干備用。2.小鼠急性毒性實(shí)驗(yàn):半數(shù)致死量法,將小鼠分為空白對照組,AHV-PI1-AHV-PI5組,空白對照組為生理鹽水溶液,AHV-PI組分別加入9.0、10.7、12.7、15.1、18.0mg/kg的AHV-PI溶液,將小鼠動物體溫計(jì)塞入小鼠肛門內(nèi)待小鼠平穩(wěn)后測量小鼠基礎(chǔ)體溫,然后腹腔注射配制好的溶液,觀察并記錄小鼠精神狀況和體溫變化,死亡小鼠記錄死亡時(shí)間后立即取出小鼠心肝肺等全身臟器用10%甲醛(福爾馬林)固定液固定。未死亡小鼠分別測定體溫,觀察一周后全部眼球取血測量血粘度變化,然后處死存活小鼠并解剖,取下各個(gè)臟器行HE染色,3.蛋白等電點(diǎn)和分子量測定:雙向電泳第一項(xiàng)等點(diǎn)聚焦,然后配膠行第二項(xiàng)垂直電泳,脫色后凝膠成像儀拍照分析AHV-PI等電點(diǎn)和分子量。結(jié)果:1.皖南蝮蛇毒毒素之中包含有AHV-PI組分,經(jīng)過陰陽離子交換以及分子篩以后能夠分離出體外強(qiáng)烈抑制血小板的該組分。2.皖南蝮蛇毒血小板抑制因子小鼠急性毒性作用可知,小鼠腹腔注射AHV-PI的LD50為13.44mg/kg其95%可信限為(12.72~14.16)mg/kg。3.AHV-PI腹腔注射小鼠體內(nèi)后小鼠體溫會出現(xiàn)短暫性下降2~5℃不等,在一定程度上隨著AHV-PI濃度的升高,體溫降低的越明顯,約2小時(shí)后未死亡小鼠體溫逐漸恢復(fù)正常。4.雙向電泳結(jié)果顯示蛇毒純化后組分AHV-PI經(jīng)過雙向電泳后可見分子量幾乎相同的五組電荷相差較小的蛋白質(zhì)點(diǎn)。拍照軟件分析后可得其相對分子量約為34.2KD,等電點(diǎn)約為4.94~4.98之間。結(jié)論:1.AHV-PI是一種小分子,偏酸性的蛇毒組分蛋白質(zhì)。2.根據(jù)前期該組分藥理學(xué)相關(guān)研究,有效作用范圍ED50在0.1mg/kg左右,TI=LD50/ED50,比值相差一百多倍,屬于使用范圍較為安全的活性組分。3.AHV-PI可以引起血粘度降低,機(jī)體凝血功能障礙,導(dǎo)致出血傾向加劇,其中肺組織出血是最為嚴(yán)重的毒副反應(yīng)。4.在蛇毒天然毒素毒性研究中存活動物中毒后的的體溫變化是相對有價(jià)值的預(yù)測因子�？梢宰鳛槲磥砩叨净钚越M分用藥安全重要的監(jiān)測指標(biāo)。
[Abstract]:Aim: to observe the acute toxicity of AHV-PI to mice and to explore the physicochemical properties of its components. Method 1: 1. The crude venom of Agkistrodon halys was purified by anion exchange method and molecular sieve snake venom protein purification column. After purification, the target peak AHV-PIwas screened by inhibiting platelet aggregation, and the target peak was freeze-dried. Acute toxicity test: the mice were divided into control group (AHV-PI1-AHV-PI5) by 50% lethal dose method. The blank control group was treated with normal saline solution AHV-PI (9.0mg / kg AHV-PI). The animal thermometer was inserted into the anus of the mice and the basic body temperature of the mice was measured after the mice were stabilized. Then the mice were injected intraperitoneally with the prepared solution to observe and record the changes of the mental state and body temperature of the mice. The dead mice were fixed with 10% formaldehyde (formalin) immediately after recording the time of death. The body temperature of the non-dead mice was measured and the blood viscosity was measured after a week's observation. Then the surviving mice were killed and dissected and the organs were taken out for HE staining. Protein isoelectric point and molecular weight measurement: the first isoelectric point focus of two-dimensional electrophoresis and the second vertical gel electrophoresis were used to analyze AHV-PI isoelectric point and molecular weight after decolorization by gel imager. The result is 1: 1. Agkistrodon halys venom contains AHV-PI component. After anion exchange and molecular sieve, we can isolate the component of AHV-PI, which can strongly inhibit platelets in vitro. The acute toxicity of platelet inhibitor of Agkistrodon halys venom showed that LD50 of AHV-PI was 13.44mg/kg and 95% confidence limit of AHV-PI was 14.16 mg / kg 路3.The body temperature of mice after intraperitoneal injection of AHV-PI decreased by 2 ~ 5 鈩，

本文編號：2027091